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Evidence for ?-helices in the large intracellular domain mediating modulation of the ?1-glycine receptor by ethanol and G??.

ABSTRACT: The ?1-subunit containing glycine receptors (GlyRs) is potentiated by ethanol, in part, by intracellular G?? actions. Previous studies have suggested that molecular requirements in the large intracellular domain are involved; however, the lack of structural data about this region has made it difficult to describe a detailed mechanism. Using circular dichroism and molecular modeling, we generated a full model of the ?1-GlyR, which includes the large intracellular domain and provides new information on structural requirements for allosteric modulation by ethanol and G??. The data strongly suggest the existence of an ?-helical conformation in the regions near transmembrane (TM)-3 and TM4 of the large intracellular domain. The secondary structure in the N-terminal region of the large intracellular domain near TM3 appeared critical for ethanol action, and this was tested using the homologous domain of the ?2-subunit of the GABAA receptor predicted to have little helical conformation. This region of ?2 was able to bind G?? and form a functional channel when combined with ?1-GlyR, but it was not sensitive to ethanol. Mutations in the N- and C-terminal regions introduced to replace corresponding amino acids of the ?1-GlyR sequence restored the ability to be modulated by ethanol and G??. Recovery of the sensitivity to ethanol was associated with the existence of a helical conformation similar to ?1-GlyR, thus being an essential secondary structural requirement for GlyR modulation by ethanol and G protein.

SUBMITTER: Burgos CF

PROVIDER: S-EPMC4279101 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Evidence for α-helices in the large intracellular domain mediating modulation of the α1-glycine receptor by ethanol and Gβγ.

Burgos Carlos F CF Castro Patricio A PA Mariqueo Trinidad T Bunster Marta M Guzmán Leonardo L Aguayo Luis G LG

The Journal of pharmacology and experimental therapeutics 20141022 1

The α1-subunit containing glycine receptors (GlyRs) is potentiated by ethanol, in part, by intracellular Gβγ actions. Previous studies have suggested that molecular requirements in the large intracellular domain are involved; however, the lack of structural data about this region has made it difficult to describe a detailed mechanism. Using circular dichroism and molecular modeling, we generated a full model of the α1-GlyR, which includes the large intracellular domain and provides new informati ...[more]

PMID: 25339760

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Project description:BACKGROUND:Stimulating the glycine(B) binding site on the N-methyl-d-aspartate ionotropic glutamate receptor (NMDAR) has been proposed as a novel mechanism for modulating behavioral effects of ethanol (EtOH) that are mediated via the NMDAR, including acute intoxication. Here, we pharmacologically interrogated this hypothesis in mice. METHODS:Effects of systemic injection of the glycine(B) agonist, d-serine, the GlyT-1 glycine transporter inhibitor, ALX-5407, and the glycine(B) antagonist, L-701,324, were tested for the effects on EtOH-induced ataxia, hypothermia, and loss of righting reflex (LORR) duration in C57BL/6J (B6) and 129S1/SvImJ (S1) inbred mice. Effects of the glycine(B) partial agonist, d-cycloserine (DCS), the GlyT-1 inhibitor, N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS), and the glycine(B) antagonist, 5,7-dichlorokynurenic (DCKA), on EtOH-induced LORR duration were also tested. Interaction effects on EtOH-induced LORR duration were examined via combined treatment with d-serine and ALX-5407, d-serine and MK-801, d-serine and L-701,324, as well as L-701,324 and ALX-5407, in B6 mice, and d-serine in GluN2A and PSD-95 knockout mice. The effect of dietary depletion of magnesium (Mg), an element that interacts with the glycine(B) site, was also tested. RESULTS:Neither d-serine, DCS, ALX-5407, nor NFPS significantly affected EtOH intoxication on any of the measures or strains studied. L-701,324, but not DCKA, dose-dependently potentiated the ataxia-inducing effects of EtOH and increased EtOH-induced (but not pentobarbital-induced) LORR duration. d-serine did not have interactive effects on EtOH-induced LORR duration when combined with ALX-5407. The EtOH-potentiating effects of L-701,324, but not MK-801, on LORR duration were prevented by d-serine, but not ALX-5407. Mg depletion potentiated LORR duration in B6 mice and was lethal in a large proportion of S1 mice. CONCLUSIONS:Glycine(B) site activation failed to produce the hypothesized reduction in EtOH intoxication across a range of measures and genetic strains, but blockade of the glycine(B) site potentiated EtOH intoxication. These data suggest endogenous activity at the glycine(B) opposes EtOH intoxication, but it may be difficult to pharmacologically augment this action, at least in nondependent subjects, perhaps because of physiological saturation of the glycine(B) site.

Dataset Information

Evidence for ?-helices in the large intracellular domain mediating modulation of the ?1-glycine receptor by ethanol and G??.

Publications

Evidence for α-helices in the large intracellular domain mediating modulation of the α1-glycine receptor by ethanol and Gβγ.

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